During the previous five years of this project, we achieved a fundamental understanding of the molecular basis of 20-hydroxyecdysone (20E) action in mosquito vitellogenesis. The goal of this application is to further characterize the genetic regulatory hierarchy mediating 20E action in the adult mosquito female. We cloned isoforms for several key factors of the 20E genetic hierarchy from Aedes aegypti. In this application, we will take advantage of the RNAi knockout technique, well established for mosquitoes, to systematically study the role of each isoform. Little is known about number and identities of target genes regulated by the ecdysteroid regulatory cascade during mosquito vitellogenesis. We will use the genomic microarray approach in combination with RNAi knockouts of specific genes to delineate novel genomic networks and target genes. We will then test the hypothesis that multiple regulatory pathways are integrated with the 20E regulatory cascade. Regulatory regions of 20E target effector genes, such as those encoding for yolk protein precursor, contain multiple binding sites for EcR/USP and the early gene products E74, E75, and BR (broad complex). We will study how these factors are involved in regulating the transcriptional activity of effector genes. We will also test the hypothesis that the early gene products, upon their binding to respective DMA sites, serve as transcriptional regulators that directly interact with the EcR/USP and cooperatively elicit a high level of expression of an effector gene in a 20E-dependent manner. We have shown that the competence factor FTZ-F1 is required for activation of the 20E regulatory hierarchy during vitellogenesis. In this application, we will test the hypothesis that FTZ-F1 directs stage-specific, 20E-induced gene expression by recruiting co-activator(s) for EcR/USP. We have discovered that alternative heterodimerization of USP either with the repressors HR38 and SVP or with an activator EcR regulates the cyclicity of vitellogenic response to 20E. In this application, we will examine whether EcR exists in a complex with heat shock proteins or other factors during the state of arrest. The proposed research is expected to advance significantly our knowledge concerning molecular mechanisms governing the key regulatory cascade in mosquito egg development, which in turn could be used for developing novel control methods against these most important vectors of human diseases.